JPS62502940A - ApoA1/C3 genomic polymorphism predicts atherosclerosis - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] The aoA■/C■ genome that crosses the pathogenesis of Ateυ-Mahi syndrome. The present invention relates to the use of genetic winterism for disease determination. The present invention is particularly In addition, apolipoprotein AI/CII [gene It is about using the winter nature of the complex.

Oie Jumei The morbidity and mortality rate of atherosclerosis in developed countries is higher than that of any other specific disease. It is higher than morbidity and mortality rates, and even higher than cancer. The disease affects the arterial cell walls. Manifested in the form of sterol deposits. Deposition progresses slowly, is irreversible, and occurs at a young age. It begins. Clinical pathology takes years to manifest and is very serious; i.e. If you have heart disease or stroke. Disease progression generally occurs as these clinical findings become apparent. It is thought that it started long before the

Environmental factors, as well as genetic factors, influence the process of cholesterol deposition and Early warning of the onset of deposition would at least provide a means of ameliorating the process. It is hoped that a diagnostic method will be found that will allow for the detection of cancer. Current technology is to detect cholera in serum. It relies on measuring terol or triglyceride levels. But these records Although BELL can be measured with great precision, it is not closely related to true susceptibility. They won't give it to me.

A more reliable prognostic method based on the detection of teloma lesions requires highly invasive methods. , as this is painful and also expensive.

Those who cannot be recruited at the screening stage are selected based on their family's medical history. It can only be applied to a special group of people. With these technologies Information is very sparse and slow; by the time atheroma lesions appear, treatment is not available. The most effective time has passed.

The importance of early detection means that effective but inconvenient and unattractive long-term treatment, i.e. Serum cholesterol due to strict diet or use of cholesterol-lowering drugs This is made even more poignantly clear by the fact that it is effective to reduce Ru. It is clear that the “deprivation” is justified. Otherwise, this approach will meet resistance.

The question is not what kind of treatment should be given.

To whom the treatment should be applied.

Techniques that inherently provide the utility of early detection should be evaluated if their results are evaluated. If it can be effectively linked to a disease, it is genetic analysis. personal Since the characteristics of the genome are basically determined, they can be used as signs of subsequent metabolic abnormalities. It is conceptually conceivable that genetic abnormalities such as these could be ideal early diagnostic tools. genetic test The test can be carried out as a routine work as early as the 7th week of pregnancy using current technology. It is possible to Restriction enzymes and DNA probing technology have advanced in the last 10 years. Ayumu (RFLP) can now be used. Successfully established to date Southern plot hybridization technique (Southern, E. , J Mol Biol (1975) 98: 503-517). used to treat sickle cell anemia (Kan, Y.W., et al., Proc.N. atlAcad Sci (USA) (1978) 75: 5631) ; β-Mediterranean anemia (Antonarakis, S.E., et al. Proc Natl Acad Sci (USA) (1983) 79: 137); Type II diabetes (Rotwein P, et al, 5c (1981) 213:1117); Familial growth hormone disorder (Phtllips+ J, A, +m, Banbur Reort 14. Co1d Spring Harbor Laboratory (1983 ) pp 305-315); Phenylketonuria (Woo, s, t,, c , l etal, Nature (1983) 306: 151); Huntington Inn (Gusella).

J, F., et al. Nature (1983) 306: 234 ); and hemophilia B (Gianelli, et al., Lancet ( 1984) i: 239. Grunenbaum.

et al, J C11n Invest (1984) 73: 1491 ) has become possible to make an effective diagnosis.

All of the previous success stories are due to special polymorphisms or winter forms associated with the disease or disorder in question. Based on gender identification. The predicted number of winter types in the human genome is Assuming that the probability that a certain arbitrary nucleotide is winter-type is 0.05, it is 107 digits. It has been calculated that this number is the human growth hormone.

α! - Inferred from studies of antitrypsin and β-like globin gene group loci. Ueffreys, A. J., Gen (1979) 18: 1- 10; 0ster, H,, et al, Am J Hum Gen (1 984) 36 (suppll) 150S). More than 10 million people like this Determine which types of winter types are related to specific disorders and how to discover them. Challenge yourself to come up with something. The present invention meets this challenge with respect to atherosclerosis. the same locations and relationships in the genome that are used to determine specific individual characteristics. It also relates to the identification of certain winter patterns and patterns of inheritance.

Hikarijiri Tachihadajo The present invention provides a winter-type syndrome that accurately predicts whether or not atherosclerosis will develop in the future. and other winter-like symptoms that indicate that the individual is protected from the development of the disease. provide specific information. Additional winterism as well. Apoliboprotein AI and CI [[code It has been discovered in this genomic region (apoA T/Cm 9TJ region).

These winter traits can be grouped together or in subsets to identify specific individuals. Provides identical profiles of people, thereby tracing patterns of genetic inheritance within families, and provides a method for investigating relationships between individuals. Furthermore, these winter-type traits are related to lipid metabolism. are present in the genomic sequences that regulate their presence or absence. The pattern may indicate additional unknown specific associations to the syndrome. would be possible.

Therefore, in one aspect, the present invention predicts the likelihood of the development of atherosclerosis in 9 individuals. The purpose is to devise a method, which method includes the following steps: D in approximately 4kb5' of apoliboprotein AI (apoAI) gene Whether approximately 300 bp of NA is deleted (’Xmn I/8.2” winter type) detecting; and/or 5.4 kb 5' of the apoA I gene ); and/or detecting the presence or absence of the first instance of the apocm gene. Detecting whether winter-morphism (“Pvu II polymorphism)” exists in the trontron; and/or winter type in 3.7kb5' of apoA I gene ("Xm nI/7.2 Presence or absence of “winter type” and the third intro of the apoA I gene To detect the presence or absence of winter type (“Msp I” winter type) in the population.

In other words, the present invention devises a method for detecting susceptibility to atherosclerosis. It is aimed at and includes the following steps: apoA I or p5'AI probe, or its substantially equivalent Detecting the presence or absence of hybridizing 8.2 kb Xmn I digested fragment; and and/or 2, which hybridizes with the p5'AI probe or substantially equivalent thereto; detecting the presence or absence of a 2 kb Apa I digested fragment; and/or 0 hybridizing with the apoCm probe or substantially equivalent thereof; detecting the presence or absence of a Pvu II digested fragment of 87; and/or 7 that hybridizes with the apoA I probe or its substantially equivalent , the presence or absence and detection of the 2 kb Xmn I fragment and the 1.75 kb Msp I fragment. and.

Furthermore, from another aspect, the present invention provides apoA1/CIII genetic Genetics of substances involved in lipid metabolism and transport disorders using polymorphisms in child regions It concerns the determination of the fingerprint of a person. The present invention also provides a method of the present invention. The company has promised to come up with a kit suitable for carrying out this task.

Concave plate Ω section plate Figure 1 shows an autoradio prepared using Xmn I digested DNA from 10 individuals. Durham, and the DNA was previously probed with apoA I cDNA. It had been googled.

Figure 2a shows the Xmn I restriction site around the apoA I gene and the 7.2 kb type χm. The estimated position of nl polymorphism is shown.

Figure 2 shows the estimated position of the 8.2 kb type Xmn I polymorphism.

Figure 3 shows the nine polymorphisms found in the apoA I/CI gene complex. It shows a map of sexuality.

Figure 4 shows the old ndlll in the 12.5kb EcoRI/EcoRI fragment. and 5 stl restriction sites; and detects a 300 bp deletion associated with the 8.2 kb type A 1.4 kb Sst I/)lindI [[genome probe (p5'AI) is shown.

FIG. 5 shows the precise location of several polymorphisms of the invention.

Kito Uni disaster relief…and In future descriptions, polymorphism distance from reference points and deletion length values may be precise; , when the size of the fragments is measured on gels or assessed by other experimental means, these Measurements of are subject to uncertainty, as will be understood by those skilled in the art. Generally > For a 4 kb measurement, the uncertainty range is ~0.3 kb; for shorter lengths The error is ~10%. Therefore, the deletion of “300bp” is smaller than that. It may be long or short, and may be 4kb from the apoA I gene. The interval between is also only an approximation.

A. Predicting patients with latent atherosclerosis 5 or general apoA I 　/　Cm When applying the method of the present invention to obtain a genetic “fingerprint”, standard Standard methods of DNA extraction, purification, restriction enzyme digestion, and size separation are used.

Hybridization with probes and successful hybridization ordered according to molecular weight. Methods for detecting hybridized substrates are also well known to those skilled in the art. heavy The general approach to discovery and detection of essential polymorphisms is as follows: Somatic cells 1 such as white blood cells, placental cells, cultured fibroblasts, or in the case of a fetus Extracted from amniotic fluid cells. Separate high molecular weight DNA fractions with special selected restrictions enzyme.

For example, EcoRI, BamHI, Mst I, Xn+n1, etc. Perform processing according to After digestion of high molecular weight DNA, the digestion product is or electrophoresed on an agarose gel and digested with restriction enzymes. Separate the DNA fragments into gel positions determined by the size (length) of the fragments. Ru. Next, the substances contained in the gel are filtered through a nitrocellulose filter or probe hive. by transferring to another suitable matrix for use as a redization support. Replica. Before transferring DNA fragments to a nitrocellulose filter replica or later treated with a denaturing agent such as NaOH/salt. Replying to denatured single-stranded DNA Labeling of the electrophoretic pattern of the purified product (-32p at the end) Single DNA fragment Probe with. Besides radioactivity, other labels such as fluorescent molecules can also be used. .

Fragments originating from specific regions on the genome are assayed using the selected probes. example For example, in the method of the present invention, apolipoprotein AI (apoAI) or apoA The cDNA sequence from the riboprotein Cnl (apocIII) gene sequence was used as a therefore.

Only the fragments that appear on the hybridized filter are complementary to this probe. , i.e.

Uncleaved within the genome itself or complementary apoA I or apoCm Only those fragments that were not cleaved by restriction enzyme cleavage. In other words , using a specific probe to determine which parts of the genome are proximal to the sequence corresponding to that probe. Alteration is detected.

The specific methods used for the general process described in the main text will not be understood by those skilled in the art. It is something that For example, methods for extracting DNA from somatic cells are described by Kan, Y., W., et al. al, Proc Natl Acad Sci (USA) (1978) 7 5: 5631-5635; Taylor, J, M, et al. Nature (1984) 251: 392-393, and Kan , Y, W, et al, Upper stem-J Med (1977) 297: 1080-1084. Improvements that allow rapid extraction of DNA Law also includes Law, D., G., et al., Gene (1984): 153-158. Size separation method for restriction enzyme-treated DNA fragments are also described in the above-mentioned references. Restriction enzyme digestion is generally performed by those skilled in the art. is standard technology.

performed under buffer, ionic strength, and temperature conditions determined by the specific restriction enzyme manufacturer. .

Transfer to nitrocellulose or other support and prehive with non-specific DNA Probing by redization.

Hybridization with a labeled probe also follows.

For example, as disclosed by the above-mentioned references and 5outhern+E, + (supra). This is the standard method used. The genomic segment being fingerprinted or On the contrary, the research questions conducted using the results naturally depend on the properties of the probe. Ru. Probes useful in the present invention include apoA I/CI [[selected from the gene complex]. It was discovered.

In B and H, the probe If the selected probe is close enough to the polymorphism in the genome that restriction cleavage will remove the polymorphism. DNA that is not separated from sex and is unlikely to be separated from polymorphism through crossover If complementary to the sequence, the resulting fragment pattern is diagnostic for a specific polymorphism. serves as evidence. The acceptable limit for the distance between the probe complementary region and the abundance is that Therefore, it is voluntary. Generally, the DNA sequence within 10 kb upstream or downstream of the polymorphism Probes that hybridize give good results. Sometimes restriction enzyme cleavage pattern Places the distal probe hybridization site on a polymorphism-independent fragment. Sometimes. The closer the probe is to the polymorphism, the more restriction enzymes can be used. The range becomes larger. Therefore.

As used herein, a probe that is “substantially equivalent” to a particular probe is If the same restriction enzyme is used, the DNA digests of individuals with a specific polymorphism will be identical. It hybridizes to the same fragment length. Therefore, the upstream 4 of the apoA I gene For Xmn I digestion diagnosis in individuals with kb5' deletion, apoAT and p The 5'AI probe (below) is equivalent. In Rsa I digestion, they are not equal. do not have. (Of course, the designated probe can be lengthened or shortened, or It can be modified in a trivial way by selecting a slightly shifted array.

) (margin below) Atherosclerosis is associated with impaired cholesterol metabolism, so plasma cholesterol The regulation of apoA I/plasma cholesterol, which is related to the regulation of cholesterol, is It is associated with polyprotein (HDL), which acts as a carrier of cholesterol in the bloodstream. known and therefore believed to play an important role in regulating its concentration. ApoA I is the main structural fluorescent substance of HDL. This has a molecular weight of 2 It is a 243 amino acid polypeptide with 8.000 amino acids. apoA I is) I Besides being a structural member of the DL complex.

It is also an activator of lecithin-cholesterol acyltransferase; The enzyme extracts milk by denoVo synthesis of cholesterol ester and solecithin. The lamellar surface structure of fat powder (chylomicrons) is transformed into pseudomicellar spherical HDL. There is a function to The apoA I protein is also. Recognized by HDL receptor It is also an HDL component.

Therefore, the apoAI gene region is associated with a prognosis for atherosclerosis and related disorders. It is thought to be the part of the genome where genome changes occur in humans. This gene is Apolipoprotein located 2.5 kb from the apoA I gene on the long arm of chromosome 11 It exists near the related gene encoding white matter Cm (apoCm) (C heung, P, +et al, Proc Natl Acad Sci ( USA) (1984) 81:508-511: Law, S, W,, e tal, Biochem Biohs Res Commun (1984 ) Dan 8:934-942). This genomic region is referred to here as “apoA I/C II [complex] for both apoA I and apoCm. Genomic and cDNA clones such as Karathanasis .

11:2827-2837 and other researchers.

Various apoA I and apoCn [probes have been described (reference example: Kar athanasis, s, g, + et al, (mentioned above); 5hold ers, S, C,, etal, (mentioned above)), until now there are no hardware for multi-objectivity detection. It has been used as a hybridization probe.

Karathanasis, S, et al, (Nature (1 983) 301 Near 1B-720; and 1 (1983) 3rd grade: 823-82 5)) 6. in the apoA1 coding region in one family suffering from IDL disorder. Indicates insertion of 5kb DNA. Rees.

A, et al, Lancet (1983) ±: 444-446 are officials? in the 3″ flanking region of the apoA I gene present in patients with hypertriglyceridemia. 5stl polymorphism. Seilhamer+ J, J,, et al, D NA (1984) 3:309-317 is the third int of the apoA I gene. MspI polymorphism in Ron.

Examples of probes useful in the present invention are apoA I and apocIII.

The p5"AI probe is described in detail in Section F.1 below.

Any probe substantially equivalent to a probe for a particular restriction enzyme, as described above. is used.

The relationship between the probe/enzyme combination and the results is approximately 4 kb of the apoA1 gene. The atherosclerosis-predicting polymorphism of the present invention consisting of an approximately 3 oobp deletion in 5″ can be explained using (see below). Deletion is 1 e.g. extracted cells D NA was digested with XmnI, the fragments were subjected to electrophoretic gel separation, and apoAI or It can be detected by probing with a p5'AI probe. below As explained, if no symptoms have actually appeared by then, the 8.2kb There is a high probability (probably 100%) that all people who exhibit this disease have a high risk of developing atherosclerosis. have. The pattern of Xmn I restriction sites in this region allows for apo The AI and p5''AI probes are essentially equivalent in this test.

Based on this pattern, within 10kb of the polymorphism, i.e. by Regarding digestion, there is a protein located within approximately 14 kb upstream or 4 kb downstream of the apoA I gene. One might expect that qualitatively equivalent probes would be discovered. however , these two probes can detect this polymorphism, but the polymorphism cleavage at a position close to the apoA I gene or at a polymorphism on the opposite side of the apoA I gene. For other series of restriction enzymes, the two are not equivalent.

For such restriction enzymes, the probe equivalent to p5'AI is determined by the restriction site and polymorphism. must be located between.

The probe uses α (“P”) dCTP and α (32P) dGTP to The probe is labeled with transrayon, which fragments the probe. subordinate Therefore, it is complementary only to the exon region of the gene (genome in Figures 2.3 and 4). (shown as a thick part of the intronic region), and the cDNA spanning the intronic region. A probe can be used in the method of the invention.

C. Atheroma arborosis ′*μ C01, - fat 1 upper μ' is the wing break method Associated with atherosclerosis. That is, one difference containing the 3oobp deletion mentioned above. The normal allele is ap rather than the 8.5kb fragment obtained in the "normal" case 8.2 kb Xmn I digested fragment detectable with oAI or p5'AI probes occurs. The definition of “normal” in the above sense must be related to the nature of the disease being diagnosed. It refers to a genome structure that does not cause any disorder in nine individuals. The majority of the total population It would be “normal”.

In order to investigate the relationship between genetic test results and atherosclerosis, 125 subjects were tested using XmnI and apoAI or p5'AI probes. did. The DNA collected from these 10 subjects had a 300bp deletion. , an 8.2 kb fragment was created. All of these 10 subjects had already undergone myocardial infarction. had lower levels of I (deletion associated with DL cholesterol). , Major signs of familial associated hyperlipidemia 1: Early development of severe atherosclerosis and its relationship The existence of a disease and its resulting clinical symptoms was recognized. 25 subjects are normal control healthy subjects; their DNA does not contain any deletions. won. The remaining 90 subjects (who also did not show the 8.2 kb fragment) Found to have atheromatous plaque as detected by angiography However, not all people had heart attacks. Therefore, this sample lacks Loss is present in people at high risk for atherosclerosis. of such individuals It's not like there are any factory workers. Therefore, a positive result in a test using this polymorphism The results are diagnostic and predictive.

Clearly prophylactic treatment is appropriate. (Of course, a negative test result indicates atherosclerosis. This does not mean that there is no risk of developing the disease; it is simply due to other genetic defects. or other factors predisposing the individual to atherosclerosis. It's just that. ) C82, Sonoya's Target Punishment Regarding various polymorphisms related to the apoA I / Cm 9i region, the above C , the analysis was conducted using a different subject group from the subject group considered in Section 1. child group of “atherosclerosis patients”, i.e. detected by angiography Having had a heart attack in a person who has been shown to have atheromatous plaques such as and the “control”, which was negative in this test. Divide into groups of people. Using this classification, the presence or absence of a specific polymorphism Statistical analysis can be performed to show the magnitude of the risk of atherosclerosis according to the current situation. Ru. Apa I, Xmn I/8.2°Xmn for 550 subjects I/7.2. Msp I, Pst I, Ban I and Pvu U Polymorphisms (see Table 2 below) were tested and analyzed against these criteria.

Based on the results of this analysis, Apa I, Xmn I/8.2 or Pvu Subjects with the If polymorphism are at increased risk of atherosclerosis; Those who have both nI/7.2 and MspI polymorphisms are not susceptible to this disease. It does not appear to be affected.

D-Moxibustion Other polymorphisms in the apoA I/Cm gene region were obtained and shown in the Table of Examples below. 2, along with those discussed in detail above. These polymorphic Several have been characterized with respect to their location, but all have specific limitations. Enzymatic digestion and blobbing with a specific probe or its substantial equivalent can be operationally defined as detectable by These polymorphisms Patterns of presence or absence can genetically fingerprint an individual. Because there is.

Genetic patterns between parents and offspring can be traced.

Additionally, these polymorphisms are associated with genomic regions important for lipid metabolism and transport. Therefore, the presence of one or perhaps several of these polymorphisms is associated with lipid metabolic disorders. This is thought to be related to the tendency of related symptoms. Currently, this group Elucidation of the precise correlation between the presence of specific polymorphisms and specific symptoms is still limited. Ru. However, confirming the presence or absence of these genetic modifications is General purpose and individuals and families to identify individuals at high risk for quality disorder syndrome It is useful for the purpose of obtaining knowledge of the genetic characteristics of the pattern.

A specific example of application is that the method of the present invention can detect individuals with Tanjour's disease. It can be explained by what it can do. Tanjour disease is a rare autosomal degeneration disorder, the loss of IDL (in the plasma), and the loss of IDL stored in some organs. It is characterized by an increased amount of tellyl esters. Clinically, the patient had enlarged tonsils and enlarged lungs. , and of Inherited Disease with characteristics of transient peripheral neuropathy. ase (McGra'1y-Hill, New York), 5 tanbur y.

Only 3 individuals exhibited habrotypes based on the characteristic 5 polymorphic cents. Therefore, this A 5-polymorphic subset of can be used as a predictor of disease progression.

E, kit The reagents used to apply the method of the invention to detect the appropriate polymorphisms are as follows: Assay packaged in a suitable container or support useful for carrying out the There are many components required for the assay. Contains type-related restriction enzymes and appropriate probes. Furthermore, hybridization concentration of reagents used in hybridization, prehybridization, DNA extraction, etc. A package containing a condensation solution is also included if necessary. However, especially the signs Reagents suitable for forming probes, or conveniently labeled probes, are 2 is useful in facilitating the performance of the method of the present invention.

F, fruit old spot The examples below are specific with respect to the probes exemplified and also include DNA extraction, It is special with respect to precise conditions such as probe hybridization.

These factors are intended to be illustrative and not limiting. Book When the invention relates to the detection of a specific polymorphism 2. The essential feature of the invention is the detection of a specific polymorphism. It's in the choice. For example, the fruit of Xmnl/8.2 that predicts atherosclerosis. Examples include ×mnl digestion of genomic DNA and proximal to the polymorphic site (i.e. ~, in this case <8.2 kb) complementary to the genomic sequence (in the unique region) A probe with a sequence can be used. Alternatively, other restriction enzymes can also be used to May be used with probes that hybridize particularly close to sex .

Various fixed polymorphisms may be detected using specific restriction enzymes. substantially A variety of probes can be designed for this region, equivalent to The cDNA to be detected is arbitrary. However, as will be understood by those skilled in the art, Note that the closer the lobe is to the polymorphic site, the more effective the probe will be. Must be. If not, other cleavage points may exist between the polymorphism and the probe. In addition, the blobbing site may also undergo crossover during the replication period, resulting in a polymorphic region. It will be separated from the rank.

F.1. Kotorokudan■Nawate White blood cells were obtained from fresh mortar collected from each subject, and high molecular weight genomic DNA was extracted from raw , D. J., et al., Gene (1984) 28: 153 158 It was isolated according to the method of

The high-molecular-weight DNA was divided into parts, each of which was purchased from the supplier (New Engla. nd Biolabs and Bethesda Re5earch Labor one of the various restriction enzymes under conditions recommended by the completely digested by Digestion products were diluted with 30mM NaHzPOa, 36m Horizontal agarose scale 7 in M Tris, 1 mM EDTA, pH 7. L/Medium T: Applied to electrophoresis. After electrophoresis, the DNA fragments were diluted with 0.5M N Denatured in aOH/1.5M NaC1 for 2 x 10 minutes at Koshiza, and 1M acetic acid solution. Neutralize with ammonium (pH 7.2) for 2 x 10 minutes and leave the nitrocellulose overnight. (Schleicher and 5chuell). fill 2x5SC (Ix5SC is 0.15M NaC1,0,015M Wash in sodium enoate, p) 17.4) and heat in vacuum at 80°C for 2 hours. Next, 5x Denhardt's solution (1x Denhardt's solution is Ficoll, polyvinyl pylori 5 5SPE (IX5SPE is 10mM sodium phosphate (pH 7,4), 0 ．． 18M NaCl and lmM EDTA).

40% (v/v) formamide and 250 μg/− of shear-modified salmon semen D 5 hours in a plastic bank using a solution containing NA at 0.3 mE/aa. Perform inter-prehybridization.

And in the same bag, 5X5SPE, IX Denhardt's solution, 40% (v/v ) formamide, shear-denatured with 10% dextran sulfate and 100 μg/- Hybridize overnight using salmon semen DNA at 0.1 ml/crA. the appropriate 32P-labeled probe 1100n/ as described below. Mix with bag (containing 1-2 filters). Prehybridization and hybridization was performed at 42°C.

The filters were then incubated in 2×SSC at room temperature and 2×SSC at 65°C.

Washed twice in 1x Denhar Bti solution. Hybridize to 3zP-labeled probe The extracted DNA sequences were transferred to XAR-5 film (Kodak) and Cronex reinforced Using a screen (Dupont), at -70°C for 18 hours, ~2 days. The images were visualized by autoradiography.

° Three specific probes, namely apo^I probe, apocI [I pro ApoA I probe and p5'AI probe were used in the following example: apoA I probe is described by Seilhamer+ J, J, + et al. (supra) 600 cleaved with Avan to remove the terminal A/T tail as described. It is a bp cDNA fragment. this probe.

BRL Nick Translation Kit (Bethesda Re5earch The presence of non-activated dATP and dTTP was determined using Bottom, α (“P”) dGTP and cx (”P) dCTP (800Ci/mm Amersham Corporation) Under the condition, 2.0 to 5. The probe was labeled with an activity of OX10”cpm/μg. Immediately before the hybridization step, incubate for 5 minutes in a boiling water bath and cool. Denatured by rapid cooling with ice water.

(Margin below) apocI [[Probe Levy-Wilson, B., et al. 480 bP S described in DNA (1984) 3: 359-364 This is a phI/Pvu 11 partial cDNA fragment. This is BpoA 1 pro It was labeled in the same way as the tube.

The p5'AI probe was prepared as follows: 12.5 kb EcoRI/ The EcoRI fragment was added to the 6kb of the 5th sequence of the apoAI gene and the complete apoAI residue. Gene + apoA Genetic region between gene and apoCm gene 2.6 A human gene containing 1.6 kb of the kb and apocI genes in this order. It was isolated from a gene library (see Figure 4).

This fragment was digested with old ndlI and 5stI, and the smaller fragment was isolated. this Most of their small fragments contain repetitive sequences. 1 obtained by this digestion, 4kb SstI/HindI [l fragment is hybridization probe It has a specific sequence suitable for p5'AI, and was named p5'AI. Do this the same way as above Labeled. The p5'AI probe is 4kb5° of the apoAI gene. Hybridizes near the atherosclerosis deletion polymorphism listed below.

Therefore, restriction endonucleases other than XmnI can be used to detect this deletion. some can be more easily separated on an agarose gel. For visualization, use apoA I or p5''AI in the procedure of item A. When used as a probe and digested with genomic DNA Z pattern was discovered. Figure 1 shows Xmn I-digested DNA from 10 subjects. Shows the autoradiogram when probing with poA I probe. . As can be seen from these results, most of the tested subjects were homozygous and 8 ．． Only a 5kb DNA fragment was produced. One subject (lane 3) scored 8.2/8. One person (lane 2) was heterozygous for the 5kb genotype and had the 7.2/8.5kb gene. one person (lane 5) was homozygous for the 7.2 kb genotype. Ta.

These two abundant sites in relation to the probe sequence complementary to the apoA T gene The position of the polymorphism is determined according to the position of the polymorphism. It was determined by treating DNA with an enzyme. This determination method is based on relevant restriction sites. For better understanding, refer to Figures 2a and b, which show diagrams of related gene sequences along with the location of can do.

In Figure 2a, the expanse of DNA that is expected to yield an 8.5 kb fragment upon XmnI digestion is shown. The gap is shown. If polymorphism exists, a 7.2kb fragment will be generated, so the two diagrams are There is another Xmn1 site at the position indicated as “a1→” or “2→” It must be.

These two possibilities suggest that in addition to XmnI, DNA from homozygotes for ApaI i [Distinguished by digesting the printed matter. If another Xmn I site is Given this position, the 1.4 kb fragment could arise from an allele containing the polymorphism. is expected. If the new site is position 1, then 2, 5 relative to the “normal” sequence Only kb fragments would be expected. Plot hybridization experiments However, only a 2.5 kb fragment was found even in subjects with the polymorphism. It was. Therefore, the polymorphism is at the position of arrow 1, or about 3.7 of the apoA I gene. kb upstream.

A similar analysis of the polymorphism resulting in the 8.2 kb fragment is shown in Figure 2.

New Xmn 1 part is either marked “1-” or “2-e” It must be the location. If the DNA was further digested with HindII, this The positional polymorphism is 3,04 kb thought to be produced by the normal allele. In addition to the fragment, a 2.64 kb DNA fragment will be generated. The result is a normal Since only a 3.04 kb fragment of the gene was discovered, the new Xmn I site The same position indicated by the arrow is approximately 4 kb upstream of the apoA I gene. I came to this conclusion. The presence of this site is due to an approximately 300 bp deletion at this position. It is created. The frequency of this deletion is calculated based on 125 subjects to be 0. It is 03.

Considering FIG. 1 again, lanes 1, 4 . and 6-10 is 8.5kb D Only a single band corresponding to the NA fragment is shown. Contains only 7.2kb fragment Lane 5 therefore corresponds to the modification at 3.7 kb 5' of the apoA I gene. Represents homozygous subjects.

Lane 2 is related to the same polymorphism as both the 8.5 and 7.2 kb fragments are present. represents a subject with a heterozygous condition. Lane 3 is 8.5kb and 8.2kb against the atherosclerosis deletion polymorphism, as indicated by the presence of both fragments. A heterozygous subject is shown.

A 300 bp deletion 4 kb upstream involved in the formation of an 8.2 kb fragment by Xmn I digestion. Using a hybridized p5'AI probe very close to the location of the loss Further restriction enzymes can be used to detect the presence of polymorphisms. . The fragments resulting from the use of these enzymes are of course the fragments obtained by Xmn I digestion. The length is different from the piece. Table 1 below shows the coverage of heterozygotes for atherosclerosis polymorphisms. The DNA obtained from the experimenter was digested with another set of enzymes and then probed with the p5'AI probe. The results are shown below. The table shows fragments related to normal genotype. The lengths and fragment lengths obtained for polymorphisms are shown.

rainbow '0 method apo I for treating atheroma p5'A　I　Xmn　I　8.5　8.2p5'A　I　Apa　I　3.5 3.2p5'A I Bgl 1 6.2 5.9p5' A I BstE II 2.5 2.1p5'A I MSI) I 4.0 5.9p5'A I PvuII 1.9 1.6p5'A I Rsa I 3.5 3.2 p5'A　I　Stu　I　5.8　5.5p5'A　I　Taq　I　5.2 9.1 The presence of a polymorphism (or Results shown in 1) are significantly correlated with atherosclerosis. As mentioned above, game All individuals with this polymorphism in Nomu are susceptible to atherosclerosis. However, the patient is showing severe urinary symptoms. However, the reverse is not true: Ate Roman sclerosis can occur even in people who do not have this genetic abnormality . None of the subjects tested were homozygous for this genetic trait: That is, they all had alleles that produced "normal" 8.5 kb fragments.

Other polymorphisms have been discovered that are associated in some way with the risk of developing atherosclerosis. Ru. to determine these polymorphisms.

positive findings regarding atherosclerotic plaque formation as determined by angiography; Gender and negative results were used as criteria to construct the control and patient groups. heart This assay detects plaque regardless of whether you have had a heart attack or not. Those who indicated were classified as “patients.” Those with negative test results were defined as “control 1; All of these people had never had a heart attack.

In interpreting this result, the standard x squared The test was used. Significance level is the level of significance that indicates that the results obtained even if the number of subjects is increased. It represents the left and probability of never being disturbed. For example, if the value is lower than 0.05 The level of concern may be the same if additional or more subjects are tested. means that the probability of giving a result is greater than 95%; a significance level of 0.10 is a test that would give different results if a larger or different sample were tested. This means that there is one chance out of ten.

The finding is that people with the polymorphism are more likely to develop the disease than people without the polymorphism. It is explained in terms of relative risk.

These “relative incidence” values are based on Wolf, B. + Ann Hum Gene Calculated according to t (1955) 19:251. Also applicable to the following assays: As shown, the relative incidence is calculated by the following formula: PP　X　CN PN　X　CP here.

pp is the number of patients with the polymorphism; PN is the number of patients without the polymorphism; cp is the number of control subjects with the polymorphism; CN is the number of control subjects without the polymorphism.

If the value calculated by this formula is significantly greater than 1.

Individuals with the polymorphism have been shown to be at greater risk of contracting the disease. less than 1 A low value indicates that there is no risk of this disease.

By applying this analysis, Apa I, Xmn I/8.2 and Pvun polymorphism correlates with increased risk of atherosclerosis.

People with both the Xmn I/7.2 and Msp I polymorphisms are at significantly lower risk. won. The respective data are as follows: for Apa I polymorphism, 1 90 of the 36 patients, or 66%, had this polymorphism, whereas the control group In the loop, 26 out of 44 people, or 59%, showed it. With this polymorphism The relative risk of atherosclerosis for people with This was 1.35 times higher than that of asexual people, and the significant difference was 0.014.

When this criterion is applied to the Xmn I/8.2 deletion polymorphism.

16 of 113 patients, or 14%, had this polymorphism, compared to controls. 4 out of 44 people, or 9%. Applying the formula, the polymorphic person The disease risk rate increased by 1.7 times.

The significant difference was 0.14.

Regarding Pvun polymorphism, 62 out of 158 patients or 39% had polymorphism. Among the controls, only 8 out of 62 people, or 13%.

As a result, the risk rate increased by 4.4 times for people with the polymorphism, with a significant difference of 0.05. Ta.

Xmn1/7.2 and Msp I polymorphisms have relative disease incidence rates of 0.5 and 0.8, respectively. This disease seems to be slightly less likely to occur.

However, the significant differences were 0.3 and 1.0, respectively. however.

If the Xmn I/7.2 and Msp I polymorphisms are combined as a habrotype, , the relative incidence is 0.25, and an individual with both polymorphisms has It has been shown that they are 4 times less susceptible to atherosclerosis. Statistically significant difference in this value was 0.03.

These values indicate that 48 out of 188 patients, or 25%, also had the Xmn1/7.2 polymorphism. 24 out of 48 controls, or 50%, had it, and 26 out of 162 patients, or 50%, had it. 16% had the Msp I polymorphism, and 10 of the 54 controls, or 18%, had this This is based on the finding that

F, 31 length■Other■Moxibustion type Deletions linked to atherosclerosis and heart attacks, and the atheroma outlined above In addition to polymorphisms predictive of sclerosis, many other polymorphisms are associated with apoA I/Cm gene duplication. It is related to merging. One of all these polymorphisms in the apoA1/Cnl region The general pattern is shown in FIG. 3 and in Table 2.

(The apoAI/Cm complex was screened with a number of enzymes that did not produce polymorphisms.

In particular, a 600 bp partial apoA I cDNA probe was used in the procedure in item A. If the DNA is digested with any of the following enzymes, no polymorphisms will be found. Not possible: Apa I, Ava I, Ava II.

BamHI, Bcll, Bgll, BglII, BstNI, EcoRT, E coRV.

HaelIl, 1(incI[, Hinfl, 1(phI, NciI, Nco l, PvuIr.

Rsa I, Sca I, 5crFI, Stu I, Taq I and and TthlII [.

When using p5'AI probe, Aval, AvaII, BamHI.

BanI, BanI[, BclI, BglII, BstNI, EcoRI, EcoRV, Haem.

HincIn, HinfI, Hphl, Ncil, Pstl, 5caI and No polymorphism was obtained for 5stI.

If apoCm probes are used, AvaII, BglI, BstE Il.

Hinfl, Mspl, Ncol, Pstl, RsaI, 5tul and Tag No polymorphisms were discovered using 1. ) (margin below) Jl-100% 1st bait"- 2” upper knee engraving (Contents (no change) probe ■-draft Nisei-e skin piece moxibustion type Seikatsupan Kouriou p5'A I Apa I 3.5 2.2 0.5apoA I Hg1AI O, 840,940,01apoA I Msp I 1.0 8.0.67 1.75 0.12apoA I Ban II O,410, 470,015apoA I Pst I 2.1 3.2 0.07apoA I Sst I 4.2 3.2 0.12apoCm Pvu U 1.0 0.87 0.3 Restrictions on a specific individual regarding a series of polymorphisms as shown in Table 2 By testing with enzymes and probes, we are able to identify genes involved in lipid transport. Obtain an individual's genetic characteristics.

Determining this genetic fingerprint specifies familial relationships and inheritance patterns It becomes a means to do so. This pattern determines parentage and the development of genetically related disorders. It is useful in tracking disease onset and predicting the occurrence of lipid transport/metabolic disorders.

For example, in particular, three people known to have Tanjour's disease should not be tested. A hablotyb (a polymorphic type) that was not found in any of the 125 people tested. are doing. This hablotype has each of the indicated polymorphisms as shown below. It occurs depending on whether it is present (+) or absent (-) in the chromosome.

The polymorphism names are shown in Table 2.

+/--/--/--/--/--/-F, 4. Di-Al/CI[r array of Table 2 is a summary of 9 polymorphisms in the apoAI/Cm region. The polymorphisms listed in the table The apoA I/CII shown in Figure 5 [accurately in the sequenced part of the gene region] It is located in The sequence is as referenced in Figure 5, and the apocIII gene The positive sense strand (meaningful strand) of the child begins approximately 180 bp upstream of the 5' end It continues almost to the 5th end of the AI gene. As shown in Figures 3 and 4 .

apoA I and apoCII [coding sequences are read in opposite directions. therefore.

The position given in the apoA I gene by the reference sequence is relative to the negative sense strand. The number increases from the downstream end of the gene.

These precisely determined positions are shown in FIG. The number in the title is ap Distance in bp from the above sequence starting approximately 180 bp upstream of oCI.

The general notation for abundant positive sites is as follows: Pvu II: G-C conversion 5 approximately 160 bp from the transcription start point of apocn[ stI/ Ban U,: C-G conversion of about 120 bp from the transcription end point of apoCm; B ann: Approximately 360 bp from the transcription end point of apoA I.

MspI: Approximately 750 bp from the start of apoAI transcription.

Era Some of the approximately 10 million polymorphisms that exist in the human genome.

- It was shown to be useful in predicting the risk of atherosclerosis.

These polymorphisms were determined by digesting the subject's genomic DNA with specific restriction enzymes and recording them here. Fragments of predictable size obtained by probing with the specific DNA sequences described. It can be detected as a piece. For early diagnosis of individuals at risk of atherosclerosis The availability of this tool allows for therapeutic measures to prevent the fatal symptoms of this disease. It can be applied at an early stage.

1 2 3 4 5 6 7 8 9 IQ5・3" Xmn1 8.5kb.

Apal -, -------, -6, -Procedure auxiliary device (method) August 28, 1986 1.Display of the incident PCT/1Js86100773 2. Name of the invention apoA I/CI predicting atherosclerosis [[Genome polymorphism 36 person who makes correction Relationship to the incident: Patent applicant Address: California, United States 94043 Mountain View, Bay Shore Parkway name Biotechnology Research Partners.

limited Representative: Newman, John H.

Nationality United States 4. Agent Address: 6-1-2-5 Nishitenma, Kita-ku, Osaka, Osaka 530, Date of amendment order August 25, 1986 (shipment date) 6. Subject of correction Translated statement 7. Contents of correction Please engrave page 30 of the translated statement on appropriate paper and submit it as shown in the attached sheet.

Country W? , research report 1++Iemaia+vlAap11calloaNa PcT, Us86. o opu71

Claims (11)

[Claims] 1. A method for predicting the development of atherosclerosis in human subjects, the method comprising: a.p. It is a deletion of an approximately 300 bp segment of DNA in approximately 4 kb 5' of the oAI gene. A method comprising detecting the presence or absence of the XmnI/8.2'' polymorphism. 2. Diagnostic long DNA fragment of human genomic DNA digested with restriction endonucleases The method of claim 1 comprising detecting the presence or absence of Therefore, if the fragment is hybridized with the p5'AI probe or a probe substantially equivalent thereto, How to Predize. 3. The combination of the restriction endonuclease and the diagnostic long DNA fragment is mnI and 8.2kb; ApaI and 3.2kb; BgIII and 5.9kb; Bst EII and 2.1kb; MspI and 5.9kb; PvuII and 1.6kb; Rsa Consisting of I and 3.2kb; StuI and 5.5kb; and TaqI and 9.1kb. 3. The method of claim 2, wherein the method is selected from the group. 4. A method for predicting the development of atherosclerosis in a human subject, the method comprising: “ApaI” polymorphism in the 5.4kb 5′ of the gene; and apoCIII inheritance Selected from the group consisting of the “PvuII” polymorphism in the first intron of the child A method comprising detecting the presence or absence of a polymorphism. 5. 2.2 kb DNA fragment of human genome digested with ApaI or PvuII to detect the presence or absence of a 0.87 kb DNA fragment of the human genome digested with 4. The method of claim 4, wherein the 2.2 kb fragment comprises P5 'Hyperidize with the AI probe or a probe substantially equivalent thereto, and The 0.87 kb fragment is an apoCIII probe or a substantially equivalent probe. 5. The method according to claim 4, wherein the method is hybridized with a tube. 6. It is a method for predicting the risk of developing atherosclerosis in human subjects. Therefore, the “XmnI/7.2” polymorphism in the 3.7 kb 5′ of the apoAI gene, and the presence of the “MspI” polymorphism within the third intron of the apoAI gene or The method encompasses detecting absence. 7. A 7.2 kb DNA fragment of the human genome digested with XmnI and digested with MspI. to detect the presence or absence of a 1.75 kb DNA fragment of the transformed human genome. 7. The method of claim 6, wherein the 7.2 kb fragment is apoA Hybridize with I or P5'AI probe or substantially equivalent probe. and the 1.75 kb fragment is apoAI probe or substantially equivalent thereof. 7. The method according to claim 6, wherein the method is hybridized with a probe. 8. Methods for detecting human subjects prone to disorders related to lipid transport and metabolism and one or more polymorphisms selected from the group consisting of the following polymorphisms: Methods involving detecting the presence or absence of gender: “ApaI” polymorphism in 5.4kb 5′ of apoAI gene; apoAI gene "XmnI/8.2" polymorphism in 4kb5' of offspring; 3.7 of apoAI gene “XmnI/7.2” polymorphism in kb5′; 3rd exon of apoAI gene “HgiAI” polymorphism within; “HgiAI” polymorphism within the third intron of the apoAI gene MspI” polymorphism; “BanII” polymorphism within the 4th exon of the apoAI gene Type: “PstI” polymorphism in 0.3 kb 3′ of apoAI gene; and a “PvuII” polymorphism within the first intron of the poCIII gene. 9. Each modification is detected in the total human genomic DNA by the following: Method according to claim 8: Digestion with ApaI and p5'AI probe. Detection of 2.2 kb fragment using Xm or substantially equivalent probe; Digestion with nI and p5'AI or substantially equivalent thereof, or Detection of the 8.2 kb fragment using aPoAI or substantially equivalent; Digestion with XmnI and p5'AI or substantially equivalent thereof, or detection of the 7.2 kb fragment using aPoAI or substantially equivalent. Out; Digestion with HgiAI and apoAI probe or substantially equivalent Detection of the 0.94 kb fragment using MspI digestion and aPoA Detection of the 1.75 kb fragment using the I probe or its substantially equivalent Digestion with BanII and apoAI probe or substantially equivalent Detection of a 0.47 kb fragment using PstI; digestion with PstI, and apo Detection of 3.2 kb fragment using AI probe or substantially equivalent ; and digestion with PvuII, and apoCIII probe or reaction with it. Detection of 0.87 kb fragment using qualitative equivalent. 10. A method for genetically identifying an individual, comprising a series of selected digests of: including determining the presence or absence of polymorphisms in genomic DNA digests. Method: Probing with an apoAI probe or substantially equivalent. The digested product contained XmnI, BanII, HgiAI, and MspI, respectively. and PstI, or those obtained by digestion with PstI, or a subset thereof. ; Digest probed with p5'AI probe or substantially equivalent thereof The digested products were ApaI, XmnI, BgII, BstEII, Msp I, PvuII, RsaI, StuI and TagI, or a subset thereof. obtained by digestion with Depletion probed with apoCIII probe or substantially equivalent. and the digested product contains BanII, SstI and PvuII, respectively. obtained by digestion with a subset of these. 11. A reagent kit useful for carrying out the methods of claims 1 to 10, comprising: paI, XmnI, HgiAI, MspI, BanII, SstI, PVuII , BglI, BstEII, RsaI, StuI and TagI. at least one suitable restriction enzyme and an apoAI gene probe or p5'AI probe or substantially equivalent thereof, and apoCIII probe or substantially equivalent thereof. A kit containing at least one probe.